Replicative Hybrid of T4 Bacteriophage DNA

Hybrid density replicative T4 DNA was isolated from CsCl, sheared, and reanalyzed in CsCl. The results rule out a branched model for T4 DNA replication and confirm that T4 DNA replicates to a conventional, semiconservative, colinear hybrid.     

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.     

Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

Marker-Dependent Recombination in T4 Bacteriophage. IV. Recombinational Effects of Antimutator T4 DNA Polymerase

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3' -> 5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49) -> 3'->5' exonuclease (gp43) -> DNA polymerase (gp43) -> DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.     

Replication and Recombination in Ligase-Deficient rII Bacteriophage T4D

Deoxyribonucleic acid replication and genetic recombination were investigated after infection of Escherichia coli with ligase-deficient rII bacteriophage T4D. The major observations are: (i) deoxyribonucleic acid synthesis is discontinuous, (ii) the discontinuities are more slowly repaired than in wild-type infection, (iii) host ligase is required for viability, and (iv) genetic recombination is increased.     

Replicative Intermediates of Bacteriophage T7 Deoxyribonucleic Acid

After infection with bacteriophage T7, parental and newly synthesized deoxyribonucleic acid (DNA) exhibit an extremely fast sedimentation rate in neutral sucrose gradients. This fast-sedimenting component (intermediate I) has a sedimentation constant of about 1,500S and contains T7 DNA as determined by DNA-DNA hybridization experiments. Pulse-chase experiments indicate that the fast-sedimenting material is metabolically active and serves as a precursor to the formation of T7 DNA. Intermediate I contains about 2.5 to 7% of the total ³H-labeled protein formed between 3 and 9.5 min after T7 infection. Treatment of intermediate I with Pronase results in the release of the DNA from the complex. At early times after infection, a second intermediate (intermediate II) can be detected which contains both parental and newly synthesized DNA sedimenting slower than intermediate I but 2 to 3 times as fast as mature T7 DNA. Intermediates I and II containing parental DNA are formed after infection of the nonpermissive host with an amber mutant in gene 1, a gene whose expression is necessary for the synthesis of most T7 proteins. The two intermediates are also observed when infection with T7 wild type is carried out in the presence of chloramphenicol.     

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.     

Region-Specific Recombination in Phage T4. III. the Effect of Host Mutations on Glucosylation-Dependent Recombination in Bacteriophage T4d

Mutations in the Escherichia coli genes recK, recL and (probably) uvrE and polA increase special (glucosylation-dependent), but not general recombination in bactriophage T4D.     

Bacteriophage T4 gp2 Interferes with Cell Viability and with Bacteriophage Lambda Red Recombination

The T4 head protein, gp2, promotes head-tail joining during phage morphogenesis and is also incorporated into the phage head. It protects the injected DNA from degradation by exonuclease V during the subsequent infection. In this study, we show that recombinant gp2, a very basic protein, rapidly kills the cells in which it is expressed. To further illustrate the protectiveness of gp2 for DNA termini, we compare the effect of gp2 expression on Red-mediated and Int-mediated recombination. Red-mediated recombination is nonspecific and requires the transient formation of double-stranded DNA termini. Int-mediated recombination, on the other hand, is site specific and does not require chromosomal termini. Red-mediated recombination is inhibited to a much greater extent than is Int-mediated recombination. We conclude from the results of these physiological and genetic experiments that T4 gp2 expression, like Mu Gam expression, kills bacteria by binding to double-stranded DNA termini, the most likely mode for its protection of entering phage DNA from exonuclease V.     

Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.     

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3′-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.     

Role of Gene 52 in Bacteriophage T4 DNA Synthesis

In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.     

High Negative Interference and Recombination in Bacteriophage T5

The process of close recombinant formation in bacteriophage T5 crosses has been studied by examining the structure of internal heterozygotes (HETs), the immediate products of recombination events. The T5 system was chosen because it permits the study of internal heterozygotes exclusively, thus avoiding the ambiguities inherent in previous studies with T4. The heterozygotes were obtained by the nonselective screening of progeny phage in a prematurely lysed sample from an eight-factor cross. The molecular structure of each HET was inferred from the strand genotypes displayed among its progeny. This investigation presents unequivocal evidence that both overlap and insertion HETs are intermediates in recombinant formation and that insertion HETs are a significant source of close double recombinants. There is evidence suggesting that mismatch repair of overlap HETs could be the source of close triple exchanges. Thus, a significant part, and perhaps all, of the high negative interference for close-marker recombination observed in this system is a direct consequence of the fine structure of the recombinational intermediates. These findings are compatible with recombination models proposed by others, in which a single branched intermediate can give rise to HETs of both the overlap and insertion types.     

Marker-Dependent Recombination in T4 Bacteriophage. III. Structural Prerequisites for Marker Discrimination

Distance- as well as marker-dependence of genetic recombination of bacteriophage T4 was studied in crosses between rIIB mutants with known base sequences. The notion of a "basic recombination," which is the recombination within distances shorter than hybrid DNA length in the absence of mismatch repair and any marker effects, was substantiated. The basic recombination frequency per base pair can serve as an objective parameter (natural constant) of general recombination reflecting its intensity. Comparative studies of the recombination properties of rIIB mutants with various sequence changes in the mutated sites showed that the main factor determining the probability of mismatch repair in recombination heteroduplexes is the length of a continuous heterologous region. A run of A:T pairs immediately adjoining the mismatch appears to stimulate its repair. In the case of mismatches with DNA strands of unequal length, formed by frameshift mutations, the repair is asymmetric, the longer strand (bulge) being preferentially removed. A pathway for mismatch repair including sequential action of endonuclease VII (gp49)----3'----5' exonuclease (gp43)----DNA polymerase (gp43)----DNA ligase (gp30) was proposed. A possible identity of the recombinational mismatch repair mechanism to that operating to produce mutations via sequence conversion is discussed.     

The Evolution of Epistasis and the Advantage of Recombination in Populations of Bacteriophage T4

Experiments reported here test two hypotheses about the evolution of recombination: first, the Fisher-Muller concept that sexual organisms respond to selection more rapidly than do asexual ones, and second, that epistasis is more likely to evolve in the absence of recombination. Populations of bacteriophage T4 were selected by the drug proflavine in discrete generations and the change in mean population fitness was monitored. Three separate selection series yielded results supporting the Fisher-Muller hypothesis. The amount of epistasis evolved was measured by partitioning the T4 map into regions and comparing the sum of the proflavine resistances of each region with the resistance of the whole. Significantly more interactions were found in phage isolated from the populations with lower total recombination than in those from populations with higher recombination. The degree to which these experiments fit preconceived notions about natural selection suggests that microorganisms may be advantageously used in other population genetics experiments.     

Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences

Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM^−/− cells. In addition, BLM^−/− cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM^−/− cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.The RecQ helicases, a subfamily of DNA helicases, carry out the unwinding of duplex DNA in the 3′ to 5′ direction. Homologs of RecQ have been identified in a wide range of organisms, from budding yeast to humans (reviewed in Ref. 1). There are five human RecQ family proteins: Blm, Wrn, RecQ1, RecQ4, and RecQ5. The BLM, WRN, and RECQ4 genes are mutated in Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively (1–3). A hallmark of Bloom syndrome cells is the drastic increase in the level of sister chromatid exchange (SCE),⁴ which results from homologous recombination (HR) associated with cross-over of the DNA damage caused during DNA replication (4, 5). It is therefore believed that Blm acts as an anti-recombination factor and inhibits aberrant recombination. This idea is supported by the observation that Sgs1, the yeast ortholog of Blm, facilitates the resolution of aberrant joint molecules during meiotic HR (6, 7) and following replication blockage (8).HR plays a critical role in the maintenance of genome stability by repairing DNA double-strand breaks (DSBs) and releasing replication blockages at damaged template strands (9, 10). The current model for HR-mediated DSB repair is that DSBs are processed to produce a 3′ single-stranded overhang, along which Rad51 is polymerized (11, 12). The resulting Rad51-DNA filament undergoes homology search and strand invasion into intact homologous duplex DNA, leading to the formation of the D-loop structure. DNA synthesis from the invading strand followed by dissociation from the homologous duplex DNA and subsequent re-annealing of the newly synthesized strand with the other end of the DSB completes the repair. This type of HR, referred to as synthesis-dependent strand anneal (SDSA), results in sequence transfer from the intact template sequence (donor) to the damaged DNA (recipient), and accounts for the majority of mitotic HR (11, 13). Extensive strand exchange of the D-loop, on the other hand, leads to the generation of Holliday junction (HJ) intermediates. SDSA does not cause cross-overs, whereas HR involving the Holliday junction often causes cross-overs, such as SCE and meiotic HR. An increase in the level of SCE in Bloom syndrome cells therefore supports the idea that Blm suppresses the formation of HJ as well as recombinogenic DNA lesions. This idea is supported by the biochemical evidence of the Blm-dependent resolution of Holliday junctions (14). On the other hand, in Drosophila, DmBlm is known to facilitate the repair of DSB by promoting SDSA (15, 16). However, the role of Blm in SDSA in the other higher eukaryotic cells has not been defined.BLM^−/− cells established from the chicken DT40 B lymphocyte line exhibit a marked increase in the frequency of both SCE and targeted integration (17–19), as do human Bloom syndrome cells (20, 21). In this study, using the chicken DT40 cells, we investigated the role of Blm in SDSA induced by defined DNA damage. To this end, we evaluated this type of SDSA using two phenotypic assays designed to analyze Ig gene conversion and DSB-induced gene targeting. Ig gene conversion diversifies the Ig variable (V) gene through HR during in vitro passage. This reaction is initiated by activation-induced cytidine deaminase-mediated uracil formation at the functional rearranged V-region (22–24). Uracil is converted to an abasic site, probably leading to a single-strand gap (25). This lesion in the functional rearranged VJ_λ stimulates the nonreciprocal sequence transfer of a single nucleotide to several hundred nucleotides, from an array of “pseudo-V_λ” regions (donor), located upstream from the functional rearranged VJ_λ, to the rearranged V region (recipient) (26–28) (see Fig. 1A). Because donor and recipient segments have an ∼10% sequence divergence, sequential Ig gene conversion events are able to substantially diversify Ig V segments. Ig gene conversion is raised only by SDSA without the formation of a Holliday junction. Hence, phenotypic analysis of Ig gene conversion provides a unique opportunity to selectively examine the role of Blm in activation-induced cytidine deaminase-induced SDSA. Moreover, nucleotide sequence analysis of Ig gene conversion products can evaluate the accuracy of HR. Like Ig gene conversion, DSB-induced gene targeting is mediated only by SDSA. The induction of DSBs by a rare-cutting endonuclease, I-SceI, at the endogenous locus, increases the frequency of gene targeting by 3 orders of magnitudes, and the frequency of gene targeting can be evaluated by measuring the reconstitution of a marker gene (29) (see Fig. 1B).Open in a separate windowFIGURE 1.Schematic diagram of assay systems used in this study. A, principle of the Ig gene conversion assay. The predominantly sIgM-negative DT40 clone contains a frameshift in its rearranged V-J_λ segments, which can be repaired by pseudogene-templated conversion events. The rate of Ig gene conversion can be measured in subclones by flow cytometric analysis of sIgM staining. B, phenotypic assays of Ig gene conversion and DSB-induced gene targeting. Pseudo-V genes and the targeting fragment act as donors for the rearranged V_λ segment and S2neo, respectively.We here show that the loss of Blm drastically reduces the rate of Ig gene conversion without compromising its accuracy or affecting the length of the gene conversion tracts, indicating that Blm plays a role in the promotion of SDSA. This is an unexpected result, because Blm is in fact believed to suppress general HR reactions, particularly recombination between diverged homologous sequences. To understand the function of Blm in SDSA, we analyzed the effect of heterologous sequences near a DSB site on HR-dependent DSB repair. The data demonstrate that Blm can promote SDSA when there is sequence divergence between the damaged recipient DNA and the homologous donor sequence. Thus, Blm has both positive and negative effects on HR, depending upon the type of DNA damage and the step of the HR reaction.